PROJECT SUMMARY/ABSTRACT Alcohol consumption is a major and growing contributor to the global burden of disease, and alcoholic liver disease (ALD), encompassing a spectrum of clinical conditions ranging from simple steatosis to acute alcoholic hepatitis, is a leading cause of liver disease both worldwide and in the United Sates. Despite extensive research, the pathogenesis of ALD remains poorly understood which is reflected in the paucity of therapeutic options available: outside of liver transplant only a single agent with a narrow therapeutic window is available, and this sole therapy ? corticosteroid therapy for severe alcoholic hepatitis ? was developed over 40 years ago. A longstanding hypothesis has been that the increased cellular NADH/NAD+ ratio as a consequence of ethanol metabolism directly contributes to the pathogenesis of ALD. However, the ability to directly test this hypothesis has been limited by the lack of available tools to directly manipulate the NADH/NAD+ ratio, and how compartment-specific changes in NADH/NAD+ impact the pathogenesis of ALD is unknown. This proposal aims to test this theory by using a newly-developed enzymatic tool, LbNOX, which converts NADH to NAD+, effectively regenerating NAD+ and lowering the NADH/NAD+ ratio in a compartment-specific manner. The core hypothesis of this proposal is that NAD+ regeneration will mitigate the effects of alcohol induced damage in both cultured hepatocytes and in mouse models of liver disease. In Aim 1, the impact of compartment-specific NAD+ regeneration on hepatocyte physiology will be investigated in cultured hepatocytes, and the pathogenic effects of ethanol metabolism, including reactive oxygen species production and inhibition of beta-oxidation, will be examined in the presence of LbNOX. In Aim 2, hepatic expression of LbNOX will be generated in mice using adeno-associated viral (AAV) vectors. These mice will be used in mouse models of alcohol-induced liver disease to examine how compartment-specific NAD+ regeneration impacts the development of liver pathology with alcohol intake. This work will demonstrate the precise role compartment-specific increases in NADH/NAD+ have on the development of ALD, and potentially offer a new therapeutic strategy for its treatment: promoting NAD+ regeneration. The work will additionally complement the proposed training plan, aiding the applicant on his path to independence as a physician scientist focused on the study of liver disease. .